We are studying the mechanisms responsible for incomplete penetrance of the renal agenesis phenotype of the mouse mutant limb deformity (ld). ld was originally identified in mice as an autosomal recessive syndrome consisting of completely penetrant reduction and fusion anomalies of the long bones and digits of all four limbs, and incompletely penetrant renal agenesis/dysgenesis. Several alleles of ld mutations have arisen on different genetic backgrounds, three of which are ldBri, ldIn2, and ld J. The ldBri and ldIn2 mutants have a 20-30% penetrance of renal abnormalities, whereas the ldJ mutants have a 98% penetrance. The ld gene encodes four alternatively-spliced isoforms (I-IV). Isoform IV null mice (ldG) have been generated by gene targeting. The limb and kidney phenotypes were distinguished in the null mice. ldG/ldG mice have a 6% incidence of renal agenesis, yet have perfectly normal limbs. The purpose of our study is to determine whether the variability of the penetrance of the renal phenotype of the various ld mutants is allele-specific, background-specific or a combination of the two. We have generated various combinations of compound ld heterozygotes that have been mated. The F2 offspring have been scored for limb and renal abnormalities. Our preliminary results indicate that the renal abnormalities correlate with the ld allele, thus suggesting that it is the mutation itself, not the genetic background, that governs the penetrance of renal agenesis at this locus. To further investigate the nature of these ld mutations, we plan to remove the entire ld locus by gene targeting in ES cells. The phenotype of this null allele will be compared with the phenotypes of other ld alleles. These mice will serve as the recipient for various modified ld alleles, introduced via YAC transgenesis.